An isoquinoline alkaloid is a class of a variety of compounds ranging up to 6,000 kinds, and is an important useful secondary metabolite produced by plants, containing many useful pharmaceutical agents such as morphine and berberine. However, almost of their production relies on extraction from natural products.
Morphine and codeine which are an analgesic, and benzylisoquinoline alkaloids such as berberine, palmatine, and sanguinarine which are antibacterial agents are synthesized from tyrosine via (S)-reticuline in Magnoliaceae, Ranunculaceae, Berberidaceae, Papaveraceae, and other many plant species. (S)-reticuline is a branching point intermediate in biosynthesis pathways of many types of benzylisoquinoline alkaloids. That is, (S)-reticuline is a pharmaceutically important non-narcotic alkaloid which is useful in developing an anti-malaria agent and an anti-cancer agent. However, production of an alkaloid at a large scale using plants is difficult under the strict control of secondary metabolism in plants. In addition, chemical synthesis of an alkaloid is difficult because structures of alkaloids are complicated.
The present inventors isolated and identified many alkaloid biosynthesis genes from gene analysis of Coptis cells having high alkaloid biosynthesis activity. In addition, the present inventors have developed a method for producing a novel useful product, particularly reticuline which is an important intermediate metabolite by means of metabolic engineering using these genes (Patent Document 1).
In recent years, by application of plant metabolic engineering to a trial of increasing an amount of a final product of an alkaloid biosynthesis pathway, selected plant cells have become possible to produce a metabolite at an industrially applicable amount. With the development of metabolic engineering, development of novel useful drugs, using intermediates as substrates has been desired. However, only a few examples of successful cases in plant metabolic engineering as to accumulation of metabolic intermediates have been reported.
Production of reticuline has been reported in a transgenic opium poppy plant by means of RNAi of codeinone reductase (Non-Patent Document 1), and in a transgenic California poppy cell by means of RNAi of berberine bridging enzyme (BBE) (Non-Patent Document 2). The transgenic opium poppy is effective in producing reticuline, but there are problems that an amount of the product considerably varies for every plant or cultured cell, and that growth of a plant or a cultured cell needs a long time. Knockdown of the final step in morphine biosynthesis by means of RNAi of codeinone reductase induced accumulation of reticuline, but the mechanism of this accumulation could not be explained. In a study using an antisense method in order to suppress BBE in a California poppy cell and a root culture, accumulation of reticuline was not observed (Non-Patent Documents 3 and 4).
Like this, the production of reticuline which is an important intermediate, for example, in transformants has been tried. However, in systems using plant bodies or cultured cells, there are problems that a long time is necessary for proliferating it and that, in many cases, products exist as a mixture.
Recently, some trials of re-constituting entire biosynthesis steps in vitro have been investigated in microorganism systems (Non-Patent Documents 5 and 6). The microorganism system has excellent ability in improving not only an amount but also quality of a secondary metabolite since other plant metabolites are not inherently present in the microorganism system. The microorganism system gives some advantages to in vivo conversion of chemical substances, but there is drawback in it that availability of a substrate is limited, particularly, in a plant metabolism. A combination of a microorganism enzyme gene and a plant-derived gene is promising for establishing an effective and highly productive system of a variety of compounds.
Regarding benzylisoquinoline alkaloid pathway, almost all biosynthesis genes from norcoclaurine to berberine have been isolated, and their activities have been shown in microorganism systems (Non-Patent Documents 7 and 8). Since norcoclaurine synthase (hereinafter also referred to as NCS) catalyses coupling of dopamine and 4-hydroxyphenylacetaldehyde (hereinafter also referred to as 4-HPAA) in the benzylisoquinoline alkaloid pathway, it has been revealed that (S)-reticuline is produced via (S)-norcoclaurine. (S)-norcoclaurine is then converted into coclaurine by an action of norcoclaurine 6-O-methyltransferase (hereinafter also referred to as 6OMT), coclaurine is converted into N-methylcoclaurine by an action of coclaurine-N-methyltransferase (hereinafter also referred to as CNMT), N-methylcoclaurine is converted into 3′-hydroxy-N-methylcoclaurine (hereinafter also referred to as 6-O-methyllaudanosoline) by an action of P450 hydroxylase, and 3′-hydroxy-N-methylcoclaurine is converted into (S)-reticuline by an action of 3′-hydroxy-N-methylcoclaurine-4′-O-methyltransferase (hereinafter also referred to as 4′OMT) (see FIG. 1a).
[Patent Document 1] International Publication No. WO 2005/03305
[Non-Patent Document 1] Allen, R. S. et al. RNAi-mediated replacement of morphine with the nonnarcotic alkaloid reticuline in opium poppy. Nat. Biotechnol. 22, 1559-1566 (2004)
[Non-Patent Document 2] Fujii, N., Inui, T., Iwasa, K., Morishige, T.,& Sato, F. Knockdown of berberine bridge enzyme by RNAi accumulates (S)-reticuline and activates a silent pathway in cultured California poppy cells. Transgenic Research, 16:363-375 (2007)
[Non-Patent Document 3] Park, S. U., Yu, M., & Facchini, P. J. Antisense RNA-mediated suppression of benzophenanthridine alkaloid biosynthesis in transgenic cell cultures of California poppy. Plant Physiol. 128, 696-706 (2002)
[Non-Patent Document 4] Park, S. U., Yu, M., & Facchini, P. J.
Modulation of berberine bridge enzyme levels in transgenic root cultures of California poppy alters the accumulation of benzophenanthridine alkaloids. Plant Mol. Biol. 51, 153-164 (2003)
[Non-Patent Document 5] Rathbone, D. A., & Bruce, N. C. Microbial transformation of alkaloids. Curr. Opin. Microbial. 5, 274-281 (2002)
[Non-Patent Document 6] Ro. D. K. et al. Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440, 940-943 (2006)
[Non-Patent Document 7] Minami, H. Dubouzet, E., Iwasa, K., & Sato, F. Functional analysis of norcoclaurine synthase in Coptis japonica. J. Biol. Chem. 282, 6274-6282 (2007)
[Non-Patent Document 8] Morishige, T., Tsujita, T., Yamada, Y., & Sato, F. Molecular characterization of the S-adenosyl-L-methionine: 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase involved in isoquinoline alkaloid biosynthesis in Coptis japonica. J. Biol. Chem. 275, 23398-23405 (2000)